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| Home > Publications database > Molecular tools for genome engineering of Corynebacterium glutamicum |
| Book/Dissertation / PhD Thesis | FZJ-2020-04239 |
2021
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-532-1
Please use a persistent id in citations: http://hdl.handle.net/2128/27716 urn:nbn:de:0001-2021051035
Abstract: Facing the demand for environmental friendly and sustainable production processes, microorganisms are engineered for the industrial biosynthesis of chemicals, fuels, or food and feed additives from renewable resources. However, microbial strain development is still laborious, time consuming and expensive, which constricts the transition to a more bio-based economy. Therefore, development and consistent improvement of molecular tools for genetic engineering as well as methods for the high-throughput characterization of engineered strain variants are of great importance. For this purpose, the CRISPR/ Cas12a recombineering method for $\textit{Corynebacterium glutamicum}$, a well-characterized microorganism employed in the industrial amino acid production, was refined by developing the flexible and easy to assemble crRNA delivery vector pJYScr. Targeting and editing efficiency of this new CRISPR/ Cas12a system was systematically evaluated by inserting genetic mutations proximal and distal to a selected PAM site in a genomic lacZ gene encoding for $\beta$-galactosidase. Subsequently, this improved method allowing for accelerated genome editing of $\textit{C. glutamicum}$ was applied in a strain engineering campaign aiming for improved L-glutamateefflux. For this purpose single-stranded DNA oligonucleotides targeting critical amino acid residuesin the mechanosensitive channel MscCG of $\textit{C. glutamicum}$ were used for CRISPR/ Cas12 recombineering. Several generated strain variants were characterized with regard to theirrespective L-glutamate efflux identifying new gain-of-function mutations, which improve L-glutamateexport in $\textit{C. glutamicum}$. To the same extent as fast and reliable genetic engineering, rapid identification of producing strain variants in larger libraries is a crucial step in strain development. In this respect, transcription factorbased, fluorescent biosensors are valuable tools in metabolic engineering allowing for semiquantitative determination of metabolites in single cells. However, transcriptional biosensors are often limited by intrinsic characteristics of the used native regulatory circuit. Moreover, signal saturation at low inducer concentrations typically limits their use in producer strains at advanced engineering stages, and the application of biosensors in heterologous host systems is often not possible. Therefore, a unified biosensor design was established, which allows fine-tuning of important sensor parameters and ensures a sensor response in a heterologous expression host. As a key feature of the design, the regulator activity can be controlled through modulation of the regulator gene expression level by using different (synthetic) constitutive promoters. Several biosensors based on transcriptional regulators LysG and PhdR and their cognate promoters from $\textit{C. glutamicum}$ were constructed for applications in the native host and in $\textit{Escherichia coli}$. Detailed characterization of these biosensors in liquid cultures and on the single-cell level using flow cytometry showed that the sensor design enables customization of important biosensor parameters as well as application of these sensors in two different bacterial species.
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